UO

Urate oxidase (UO, EC 1.7.3.3), also called uricase, is a peroxisomal enzyme in the purine degradation pathway catalyzing the oxidation of uric acid by molecular oxygen to an inactive and soluble metabolite, allantoin, in most mammals to prevent the accumulation of uric acid, producing 5-hydroxyisourate and hydrogen peroxide. This enzyme prevalently exists in many organisms, but it is not detected in humans and certain other primates, which might be a consequence of an inactivation of the hominoid UO gene caused by independent nonsense or frameshift mutation. Since uric acid exhibits strong anti-oxidant ability that could result in fewer free radicals and fewer instances of cancer caused by aging, it has been suggested that uric acid takes a significant role in protecting hominoids from oxidative damage and prolonging the live span. However, the lack of UO could cause an elevation in uric acid levels in the plasma, which can be fatal. UO first extracted from the fungus Aspergillus flavus, now is widely expressed in Saccharomyces cerevisiae for research and medical purposes. UO possesses a unique catalytic mechanism, where both cofactor and particular metal ion are not requisite, and the general procedure is shown blow.

Figure 1. The purine degradation pathway.

Structure

UO, primarily distributing in liver, exists as a homo-tetramer of identical subunits with four active sites located at the dimeric interfaces between its four subunits, and forms a large electron-dense paracrystalline core in many peroxisomes. UO from A. flavus consists of 301 residues with a molecular weight of 33438 daltons. Sequence analysis of UO from several organisms has indicated that 24 amino acids are conserved in UO, among which 15 are associated with the active site.

The integrated functional structure of UO is a 135-kDa homo-tetramer shaped as a barrel with height of 7 nm, an inner radius of 0.6 nm and an outer radius of 3 nm. Four identical active sites of UO have been found at dimeric interfaces formed between the monomers and a central void tunnel with unidentified function. Each monomer is made up of 301 amino acid residues, and bears two structurally equivalent domains, which are comprised of a four-strand long antiparallel beta sheet with two helices on the concave side and are termed “tunneling fold” domains, allowing UO to be engaged in the extended family of tunnel-shaped proteins. The two tunneling fold domains of each monomer could form an eight-strand long antiparallel beta sheet by gathering together, where all four helices are located at the concave side of the sheet. The dimer forms an α8β16 barrel, whose outer surface is occupy the eight helices.

Action Mechanism

UO is the first enzyme in a pathway of converting uric acid to S-(+)-allantoin. After the transformation of uric acid to 5-hydroxyisourate by UO, 5-hydroxyisourate (HIU) is then catalyzed into 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) by HIU hydrolase, and ultimately to S-(+)-allantoin through 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase (OHCU decarboxylase). HIU will spontaneously decompose into racemic allantoin without the participation of HIU hydrolase and OHCU decarboxylase.

There is a catalytic site within the active site of UO, which holds uric acid and its analogues in the same orientation and a common catalytic site, where oxygen, water, and hydrogen peroxide are sequentially actuated. This mechanism reverses hydrogen peroxide back to oxygen and successively drives hydrogen peroxide and water through the common catalytic site. Two residues, Arg 176 and Gln 228, within the active site, contribute to hydrogen-binding with the substrate uric acid as a monoanion, which is then deprotonated to a dianion to be stabilized by Arg 176 and Gln 228 of the UO. Subsequently, oxygen accepts an electron pair from the uric acid dianion and is reduced to hydrogen peroxide, which is substituted by water to conduct a nucleophilic attack on the intermediate with the purpose of producing 5-hydroxyisourate. UO could be inhibited by both cyanide and chloride ions owing to anion-π interactions between the inhibitor and the uric acid.

Figure 2. A proposed mechanism for urate oxidase. (Gabison L; et al. 2008)

Function

UO is capable of preventing the excessive building up of uric acid levels in certain organisms, including bacteria, fungi, yeast, and some mammals. Although the exact mechanism through which UO exerts its function is not illuminated yet, there is an agreement that the previously described proton transfer is involved in the catalysis.

UO is also essential in the ureide pathway, where the fixation of nitrogen happens in the root nodules of legumes, and the fixed nitrogen is then converted into metabolites. Finally, the generated metabolites are transported from the roots throughout the plant to supply the necessary nitrogen source for amino acid biosynthesis.

It has been also proposed that the loss of UO gene expression is advantageous to hominids, since uric acid is a powerful antioxidant and scavenger of singlet oxygen and radicals to protect the body from oxidative damage, thus extending lifespan and diminishing the rates of age-related cancer.

Medical Implications

Hyperuricaemia is most commonly connected with gout and malignancy in mammalians, especially those suffered with lymphoid malignancies caused by rapid cell turnover and an augmented rate of purine metabolism. UO has been developed as an efficient protein drug in the prevention and treatment of acute hyperuricaemia hyperuricemia in patients receiving chemotherapy and in those who have experienced transplantation. It shows a rapid and safe efficacy and induces a sharp reduction in plasma levels of uric acid. However, if patients lack the subsequent enzyme HIU hydroxylase in the pathway to degrade 5-hydroxyisourate into allantoin, UO therapy could be potentially harmful due to the toxic effects of HIU.

Graft-versus-host disease (GVHD) is often an attendant side effect of allogeneic hematopoietic stem cell transplantation, driven by the nuisance of donor T cells to host tissue. Uric acid could enhance T cell response, and therefore relevant clinical trials have proved that UO can be administered to suppress uric acid levels in the patient and consequently bring down the likelihood of GVHD.

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